For isolation and purification of a protein of interest from contaminants, physico-chemical properties such as a molecular size, an electric charge on the surface or solubility of said protein is utilized. A process for purification commonly used in the field of protein chemistry includes, for instance, salting out, ultrafiltration, isoelectric precipitation, electrophoresis, ion exchange chromatography, gel filtration chromatography, affinity chromatography, and the like. In case that a protein of interest must be purified from the living tissues, cells, or blood, where an enormous variety of different proteins exist, these processes need often be combined in a manifold manner. However, it is possible to provide a method for purification with much more specificity by utilizing a property commonly shared by a certain kind of protein.
By way of example, a unique method for purification using anion exchange chromatography is known wherein a divalent cation-binding protein is adsorbed to an anion exchange resin and then eluted therefrom with a divalent cation to specifically purify said divalent cation-binding protein as disclosed in Japanese patent publication No. 200180/1990. According to this method, a chelating reagent such as ethylenediaminetetraacetic acid (EDTA) is added to a solution containing a divalent cation-binding protein to first remove divalent ions. Then, the resulting solution is contacted with an anion exchange resin such as MonoQ to render the divalent cation-binding protein be adsorbed to the anion exchange resin. Finally, addition of sodium chloride and calcium chloride elutes the divalent cation-binding protein from the anion exchange resin. However, most of naturally occurring proteins are negatively charged under physiological conditions and hence numerous contaminants other than a protein of interest are preferentially adsorbed to an anion exchange resin, thus hampering efficient purification of the desired protein. Therefore, this method for purification through adsorption of a desired protein to an anion exchange resin is preferably used for a small amount of a protein solution or at an advanced stage of purification processes.
Japanese patent publication No. 258286/1995 discloses a method for purifying a calcium ion-binding, vitamin K-dependent protein by an anion exchange process wherein calcium chloride is added to a solution containing a vitamin K-dependent protein and the resulting solution is passed through an anion exchange resin to isolate the desired protein from contaminating proteins. This method, however, is disadvantageous in that a large volume of fractions containing the desired protein must be passed through and hence subsequent procedures will become troublesome especially when conducted in a large scale.
Annexin V, one of calcium ion-binding proteins, is a simple protein of about 34 kDa molecular weight bearing no sugar chain that has a physiological activity such as anti-coagulating activity, corneal epithelium-extending activity, and phospholipase A2 inhibitory activity. It is known that Annexin V distributes in a variety of tissues and secretions within the living body including human placenta (Chem. Pharma. Bull., 38, 1957-1960, 1990). Annexin V is called a calcium ion-binding protein since it has an ability to bind with a lipid membrane via calcium ions.
Annexin V has been extracted from organs of human or animals (Japanese patent publication No. 174023/1987). Nowadays, however, it can be produced in E. coli and yeast by the use of the genetic recombination technique (Japanese patent publications No. 20095/1989 and No. 219875/1991).
Annexin V has conventionally been purified, after pretreatment of an Annexin V-containing solution with precipitation, membrane filtration and centrifugation, by a combination of ammonium sulfate fractionation, anion exchange chromatography, hydrophobic chromatography and affinity chromatography (Jurgen Romisch et al., Biochem. J. 272, 223-229, 1990; T. R. Hawthorne et al., Journal of Biotechnology 36, 129-143, 1994).